The invention relates to an arrangement for nucleic acid sequencing by tunneling current analysis.
A multiplicity of methods for nucleic acid sequencing are known in the literature. They include methods of so-called sequencing-by-synthesis. In this case, a component is liberated when an appropriate nucleotide is incorporated, said component being detected by enzyme cascades. Nucleic acid sequencing in nanopores is furthermore known. It is advantageous that neither a marking of the DNA strand nor a complex reaction cascade is required in this method.
In the case of nucleic acid sequencing by nanopores, DNA strands pass through a biological or artificial (referred to as solid-state) nanopore. Individual bases of the nucleic acid strand can be analyzed as a result of a change in the pore resistance when the DNA passes through the nanopore. In this case, the DNA is passed into a conductive fluid. A voltage is applied to the fluid, with the result that an electric current flows. This current changes when different types of base (in particular guanine, cytosine, thymine, adenine) pass through the nanopore. This change is dependent on the base which passes through the pore, such that the base can be analyzed.
Alternatively, it is possible to measure a tunneling current via the pore (referred to as: sequencing-by-tunneling), wherein the tunneling current is dependent on the base situated in the pore. Tunneling current methods advantageously have a better base resolution in comparison with the measurement of the pore resistance. The latter stems from high electric field strengths within the nanopore.
These measurements are greatly dependent on the size and shape of the nanopore. Furthermore, the electrodes for applying a voltage to the nanopore have to be arranged exactly, in order to detect the tunneling current sufficiently accurately. In order to achieve a high analysis reliability of the nucleic acid sequencing by tunneling current analysis, it is desirable to produce customized nanopores with electrodes for defined applications. Most production methods for producing solid-state nanopores are based on the removal of material from a thin membrane, in a manner similar to the drilling of holes. This production is carried out in particular by electron beam-based photolithography.
It is disadvantageous that the previous arrangements of solid-state nanopores with electrodes are very complex and time-consuming to produce. A sufficiently precise arrangement of the nanopore with respect to the electrodes or a capacitor in such a way that tunneling currents can be measured with high accuracy is virtually impossible to realize technically at the present time and the production thereof is disadvantageously associated with high expenditure in terms of labor and time.